The large-scale, economic purification of proteins is an increasingly important problem for the biotechnology industry. Generally, proteins are produced by cell culture, using either eukaryotic or prokaryotic cell lines engineered to produce the protein of interest by insertion of a recombinant plasmid containing the gene for that protein. These cells must be fed with a complex growth medium, containing sugars, amino acids, and growth factors, usually supplied from preparations of animal serum. Separation of the desired protein from the mixture of compounds fed to the cells and from the by-products of the cells themselves to a purity sufficient for use as a human therapeutic poses a formidable challenge.
Procedures for purification of proteins from cell debris initially depend on the mechanism of expression for the given protein. Some proteins can be caused to be secreted directly from the cell into the surrounding growth media; others are made intracellularly. For the latter proteins, the first step of a purification process involves lysis of the cell, which can be done by a variety of methods, including mechanical shear, osmotic shock, or enzymatic treatments. Such disruption releases the entire contents of the cell into the homogenate, and in addition produces subcellular fragments that are difficult to remove due to their small size. These are generally removed by differential centrifugation or by filtration. The same problem arises, although on a smaller scale, with directly secreted proteins due to the natural death of cells and release of intracellular host cell proteins in the course of the protein production run.
Once a clarified solution containing the protein of interest without large cellular debris components has been obtained, its separation from the remaining other proteins produced by the cell is usually attempted using a combination of different chromatography techniques. These techniques separate mixtures of proteins and other impurities on the basis of their charge, degree of hydrophobicity, or size. Several different chromatography resins are available for each of these techniques, allowing accurate tailoring of the purification scheme to the particular protein involved. The essence of each of these separation methods is that proteins can be caused either to move at different rates down a long column, achieving a physical separation that increases as they pass further down the column, or to adhere selectively to the separation medium, being then differentially eluted or displaced by different solvents or displacers. In some cases, the desired protein is separated from impurities when the impurities specifically adhere to the column, and the protein of interest does not, that is, the protein of interest is present in the “flow-through”. Publications concerning protein purification include Fahrner et al., Biotechnol Genet Eng Rev. 2001; 18:301-27.
A typical large-scale purification process for antibodies is often built around the employment of immobilized protein A as the primary capture and purification step in combination with other column operations. Protein A is a cell wall protein from Staphylococcus aureas with affinity for the Fe region of IgG. For this reason it is used extensively for IgG purification. Protein A column operations in general deliver a product-related purity over 98% with most process impurities washed away in the flow-through fraction. However, there are numerous drawbacks to the use of Protein A chromatography. First, binding is usually done at a neutral to slightly basic pH and elution is usually at an acidic pH. One of the potential problems is that low pH can denature or partially denature the IgG. Because of this and the high product purity required for clinical applications, additional concentrating and purifying steps are required for separation of product-related isomers and removal of remaining amounts of host cell proteins/DNA, cell culturing impurities, leached protein A, and viruses. A compounding problem is that many of these impurities can interfere with the efficiency of downstream process operational units for isolating purified antibodies. Another main problem is price; Protein A columns are far more expensive than conventional ion exchange columns. Finally, there are numerous scenarios where Protein A chromatography is either not suitable or cost prohibitive, for example with the purification of polypeptides, antibody-like molecules, antibody fragments, and/or full antibodies purified from certain cell systems.
The nature of the present invention addresses the above identified problems and in its embodiments demonstrate an alternative purification method to those currently available in the art using a Protein A step in antibody, antibody fragment and polypeptide purification.